We intend to solve the crystal structure of cholera toxin at atomic resolution by using protein crystal diffraction methods. We have found that commercial preparations of cholera toxin are isoelectrically inhomogeneous. By purifying a single isoelectric variant, we can now grow large single crystals of cholera toxin reliably and reproducibly. We have collected good quality three-dimensional diffraction data sets from these crystals, to 3.0 alpha resolution, and are now beginning to search for heavy atom isomorphous derivatives. We plan to use the standard methods of Multiple Isomorphous Replacement to determine phases of our structure factors. At the same time, we plan to examine electron microscopic images of thin-sectioned cholera toxin crystals and construct a three-dimensional, 20 alpha resolution model of the crystal structure by combining information from several views (orientations) of the crystal. That model will define the molecular envelope, i.e., the boundary between protein and solvent regions within the crystal. This molecular envelope will then be used in procedures of real-space direct methods: molecular fivefold averaging of the B-subunits, solvent leveling, negative and positive density truncation, and entropy maximization. Such methods have been shown to improve phases that are derived from isomorphous replacement and can predict phases at higher resolution ("phase extension"). With phases for our structure factors derived from these procedures, we will calculate an electron density map that we should be able to interpret and refine to yield a correct structural model.